The mammalian target of rapamycin (mTOR) signaling network has emerged as a master regulator of essential processes in cell and developmental biology, including cell growth, proliferation, cellular differentiation, and metabolism. The multiple FDA-approved clinical applications of rapamycin attest to the central importance of mTOR in medicine. Recent evidence has also revealed a key role for mTOR signaling in modulating lifespan in addition to impacting health span. Molecular dissection of the mTOR regulatory network will facilitate development of therapeutics against aging and aging-related diseases, including cardiovascular diseases, metabolic diseases, and cancer. mTOR assembles two biochemically and functionally distinct protein complexes - mTORC1 and mTORC2, which nucleate distinct pathways and yet crosstalk extensively. The current knowledge of mTOR complex assembly is largely derived from conventional biochemical characterizations, which typically require lengthy and strenuous purification procedures that may disrupt native complexes, and offer limited insights into the stoichiometry and heterogeneity of complex formation. We (the Ha laboratory) have recently developed a single-molecule pull-down (SiMPull) method that enables rapid and sensitive analysis of protein complexes at single-complex resolution, directly from whole cell lysates. Here we propose to dissect the assembly of mTORC1 and mTORC2 employing the SiMPul approach. Stoichiometry of each component in the complexes will be determined. Regulation of the complexes by upstream signals, and potential physical crosstalk between the two complexes, will also be examined. Furthermore, we will develop a single-molecule kinase assay with the SiMPull platform, and apply this novel approach to interrogating the activities of various mTOR complexes. The proposed study is expected to decipher the assembly of both mTORC1 and mTORC2 at the single-complex level and reveal structure-function relationships in the mTOR assemblies. Knowledge to be gained from this study will be invaluable in guiding future investigation of the mTOR regulatory network and in facilitating potentially novel design of therapeutic strategies.